Interrelated involvement of the endocannabinoid/endovanilloid (TRPV1) systems and epigenetic processes in anxiety‐ and working memory impairment‐related behavioural effects of nicotine as a stressor

Abstract The addictive use of nicotine contained in tobacco is associated with stressor‐like emotional and cognitive effects such as anxiety and working memory impairment, and the involvement of epigenetic mechanisms such as histone acetylation has recently been reported. Although the precise nature of behavioural plasticity remains unclear, both anxiogenic‐ and working memory impairment‐like effects were observed in the present experimental model of mice treated with repeated subcutaneous nicotine and/or immobilization stress, and these effects were commonly attenuated by the histone deacetylase (HDAC) inhibitors that induce histone acetylation. Such HDAC inhibitor‐induced resilience was mimicked by ligands for the endocannabinoid (ECB) system, a neurotransmitter system that is closely associated with nicotine‐induced addiction‐related behaviours: the anxiogenic‐like effects were mitigated by the cannabinoid type 1 (CB1) agonist arachidonylcyclopropylamide (ACPA), whereas the working memory impairment‐like effects were mitigated by the CB1 antagonist SR 141716A. Moreover, the effects of the HDAC inhibitors were also mimicked by ligands for the endovanilloid (transient receptor potential vanilloid 1 [TRPV1]) system, a system that shares common characteristics with the ECB system: the anxiogenic‐like effects were mitigated by the TRPV1 antagonist capsazepine, whereas the working memory impairment‐like effects were mitigated by the TRPV1 agonist olvanil. Notably, the HDAC inhibitor‐induced anxiolytic‐like effects were attenuated by SR 141716A, which were further counteracted by capsazepine, whereas the working memory improvement‐like effects were attenuated by capsazepine, which were further counteracted by SR 141716A. These results suggest the contribution of interrelated control of the ECB/TRPV1 systems and epigenetic processes such as histone acetylation to novel therapeutic approaches.

observed in the present experimental model of mice treated with repeated subcutaneous nicotine and/or immobilization stress, and these effects were commonly attenuated by the histone deacetylase (HDAC) inhibitors that induce histone acetylation.
Such HDAC inhibitor-induced resilience was mimicked by ligands for the endocannabinoid (ECB) system, a neurotransmitter system that is closely associated with nicotine-induced addiction-related behaviours: the anxiogenic-like effects were mitigated by the cannabinoid type 1 (CB1) agonist arachidonylcyclopropylamide (ACPA), whereas the working memory impairment-like effects were mitigated by the CB1 antagonist SR 141716A.Moreover, the effects of the HDAC inhibitors were also mimicked by ligands for the endovanilloid (transient receptor potential vanilloid 1 [TRPV1]) system, a system that shares common characteristics with the ECB system: the anxiogenic-like effects were mitigated by the TRPV1 antagonist capsazepine, whereas the working memory impairment-like effects were mitigated by the TRPV1 agonist olvanil.Notably, the HDAC inhibitor-induced anxiolytic-like effects were attenuated by SR 141716A, which were further counteracted by capsazepine, whereas the working memory improvement-like effects were attenuated by capsazepine, which were further counteracted by SR 141716A.These results suggest the contribution of interrelated control of the ECB/TRPV1 systems and epigenetic processes such as histone acetylation to novel therapeutic approaches.

| INTRODUCTION
2][3] Chronic NC exposure results in increased emotional symptoms such as anxiety, and concurrent cognitive deficits such as impaired working memory have often been reported. 4ke NC consumed by cigarette smokers, various stressors induce anxiety as a defensive behavioural response, which is accompanied by cognitive problems. 5,6Along with "stressor-like" behavioural effects, the dysregulated stress response in the brain has also been reported as NC-induced plasticity. 7,8Moreover, in some stressor-exposed smokers, pronounced exacerbations of both emotional symptoms and cognitive deficits have been observed. 9,10On the other hand, anxiolytic, memory-improving and stress-relieving effects have also been reported depending on the smoking-related condition. 11,12With respect to such behavioural heterogeneity, the interrelated contributions of multiple mechanisms including the characteristic bimodal involvement of nAChRs, the direct molecular targets of NC, along with the antistress effects of the main NC metabolite cotinine have been suggested, [13][14][15] but the details have not been elucidated.
Epigenetic mechanisms, which regulate chromatin structure and gene expression, are associated with synaptic/circuitry function related to drug addiction and other psychiatric conditions. 16,172][23] Although epigenetic processes in the stress-related behavioural effects of NC remain insufficiently explored, a pivotal integrative role of histone acetylation in the brain has been suggested for NC-related behavioural plasticity. 24,257][28] Increasing attention has also been focused on the involvement of the ECB system in stress-related emotional and cognitive responses. 29,30[35][36][37] However, little is known about the epigenetic mechanisms associated with the ECB-TRPV1 crosstalk.In the present experimental study, the interacting influence of ECB/TRPV1 system-related ligands and histone deacetylase (HDAC) inhibitors, which induce histone acetylation, on the NC-and/or stressor-induced anxiety-and working memoryrelated behaviours, is evaluated, and therapeutic importance of the mutual control of the ECB/TRPV1 systems and epigenetic processes is discussed.

| Animals
The experiments were performed on male ICR mice (80 ± 10 days old) (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) housed in a forced-air facility, which was maintained at 23 C and 50% relative humidity, and kept on a 12-h light/dark cycle. 25,38The mice were kept individually in single transparent cages measuring 23.5 Â 16.5 Â 12 cm, and were allowed water and rodent chow ad libitum.The experiments described in this report were approved by the Kyoto University Animal Experimentation Committee, and were conducted in accordance with the "Regulations on Animal Experimentation at Kyoto University" of the institution, 39 which is based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.All efforts were made by trained personnel to minimize the pain experienced by the mice.No mice died during the experiments.
All of the observations and evaluations were performed by a trained observer who was blinded to and not informed of the treatment conditions in advance.Each experimental group comprised 10 mice, based on previously published research and preliminary data. 25,38

| Drug and stressor treatments
In the NC treatment groups, repeated subcutaneous (sc) doses of NC that caused both anxiety-and working memory impairment-like behaviours effectively in mice were selected: a single sc dose of 0.8 mg/kg was administered daily for 4 days. 25,38NC (Nacalai Tesque, Inc., Kyoto, Japan) was supplied in free-base form at 95% purity, and was freshly dissolved in saline to a volume of 5 mL/kg immediately before each administration. 25,38,40For the stressor treatment groups, repeated immobilization (IM) stress treatments in which anxiogenicand working memory impairment-like effects similar to those of the NC treatments were selected: 10 min of IM, which was induced by placing the mouse in a narrow space (diameter about 12 cm) in a vinyl bag with some breathing holes, was performed once a day for 4 days. 25,38Furthermore, to investigate the interactions between NC and IM, the behavioural alterations were examined in the NC plus IM group (NC-IM group) which received the aforementioned sc dose of NC 10 min before the IM treatment once a day for 4 days. 25,38,419][50] The doses were selected from those that induced no toxic behavioural alterations (e.g., continuous suppression of locomotor activity) by themselves at the prescribed time point even when combined administration was repeated.The drugs were dissolved and diluted using a mixed solution of dimethylsulphoxide (DMSO) plus distilled water, and were administered in a total volume of 2.5 mL/kg 60 min (drugs except for VA) or 30 min (VA) before each NC, IM, or NC-IM treatment.Furthermore, in the experiments examining the interacting role of HDAC inhibition (histone acetylation) with the ECB and/or TRPV1 system, each CB1 and/or TRPV1 ligand was used in combination with the effective HDAC inhibitors.In the HDAC inhibitor-or CB1/TRPV1 ligand-only groups, an equivolume saline vehicle was injected instead of the NC, IM, or NC-IM treatment.In the control group without any drug or stressor treatment (control group), a mixed vehicle solution of DMSO and distilled water was injected instead of the HDAC inhibitors or CB1/TRPV1 ligands, and then, an equivolume saline vehicle was injected instead of the NC, IM, or NC-IM treatment.The drug and stressor treatments and each experimental session were performed 4-8 h after the beginning (8:00 a.m.) of the light cycle.

| Y-maze test
Alterations in working memory-related behaviours were examined in the Y-maze test using a cardboard apparatus that consisted of three enclosed arms 30 Â 5 Â 15 cm (length, width, and height) that converged on an equilateral triangular center platform 6][57] After the number of spontaneous alteration performance (SAP), which was defined as the number of successive triplet entry performances into each of the three arms without any repeated entries, and the total number of entries into arms were evaluated (8-min test periods), the rate of spontaneous alteration performance (SAP rate) (%) was calculated as a parameter for the working memory-related behaviours.These evaluations were performed at the 2-h time point after the last NC, IM, or NC-IM treatment.At the beginning of each experimental session, each mouse was placed in the center platform of the maze, facing all three arms immediately before the session.

| Statistical analysis
The data were subjected to two-or three-way analysis of variance (ANOVA) for each experiment. 25,38,58With respect to the experi-  S7).
In the groups cotreated with the CB1 antagonist SR or TRPV1 agonist  S8).
Against these working memory impairment-like behavioural alterations, significant mitigating effects, that is, recoveries from the attenuated SAP rates, were observed in the NC, IM, and NC-IM groups  S8).In the groups cotreated with the CB1 agonist AC or TRPV1 antagonist CZ, as well as in each HDAC inhibitor (SB or VA)-or CB1/TRPV1 ligand (AC, SR, OL or CZ)-only group, no significant alterations in SAP rates as compared with the control group were observed under the present experimental conditions.

| Anxiety-related interacting effects between the mitigating drug (HDAC inhibitor, CB1 agonist, or TRPV1 antagonist) and CB1 antagonist with or without additional TRPV1 antagonist
Based on the data shown in Figure 1 and Table S1, interactions with Table S3D).The data in Figure 3 and Table S3 are consistent with the results of three-way ANOVA revealing statistically significant interac-  S2. (A) SB (100 mg/kg, ip) cotreatment groups (SB groups); (B) VA (300 mg/kg, ip) cotreatment groups (VA groups); (C) OL (1 mg/kg, ip) cotreatment groups (OL groups); (D) SR (1 mg/kg, ip) cotreatment groups (SR groups).**P < 0.01: significant attenuation as compared with the control group; ++P < 0.01: significant increase as compared with the NC, IM, or NC-IM group without any cotreatments; @@P < 0.01: significant attenuation as compared with the NC group without any cotreatments; $$P < 0.01: significant attenuation as compared with the IM group without any cotreatments.S7).In each HDAC inhibitor (or CB1 agonist AC) plus CB1 antagonist SR plus TRPV1 antagonist CZ-only group, no significant alterations as compared with the control group were observed for any parameter value under the present experimental conditions.

| Working memory impairment-related interacting effects between the mitigating drug (HDAC inhibitor, TRPV1 agonist, or CB1 antagonist) and TRPV1 antagonist with or without additional CB1 antagonist
Based on the data shown in Figure 2 and Table S2, interactions with the TRPV1 antagonist CZ (0.1-5 mg/kg) were examined for the most effective dose of the HDAC inhibitor SB (100 mg/kg) or VA (200 mg/kg), as well as for the most effective dose of the TRPV1 agonist OL (1 mg/kg), to investigate the interacting roles of HDAC inhibitors with the TRPV1 system.Against the "working memory improving-like" effects of SB or VA, as well as against those effects of OL, significant attenuating effects were observed for CZ (1-5 mg/kg) in the NC, IM, and NC-IM groups (Figure 5A-C and Table S5A-C).
Moreover, even against the most efficacious dose of the CB1 antagonist SR (1 mg/kg), significant attenuating effects were observed for CZ (1-5 mg/kg) in the NC, IM, and NC-IM groups (Figure 5D and Table S5D).The data of SAP rates in Figure 5 and Table S5 are S8).In each HDAC inhibitor (or TRPV1 agonist OL) plus TRPV1 antagonist CZ plus CB1 antagonist SR-only group, no significant alterations in SAP rates as compared with the control group were observed under the present experimental conditions.

| NC-and/or IM-induced anxiety-and working memory impairment-like behavioural alterations and mitigating effects of HDAC inhibitors
In the NC group receiving repeated treatments of NC, as well as in the IM group, anxiety-like behavioural alterations in the EPM test and working memory impairment-like behavioural alterations in the Y-maze test were observed, which supports the findings from previous studies. 25,38[61] In cooperation with such neural circuit mechanisms, the contribution of altered activity of stress-related neurotransmitter systems (e.g., dopaminergic system) has been reported for the anxiety-and working memory-related behavioural effects of NC. 60,[62][63][64] Regarding interactions between NC and IM in the NC-IM group, significant enhancements of both anxiogenic-and working memory  S3. (A) SB (100 mg/kg, ip) plus SR (1 mg/kg, ip) groups (SB + SR groups); (B) VA (300 mg/kg, ip) plus SR (1 mg/kg, ip) groups (VA + SR groups); (C) AC (0.2 mg/kg, ip) plus SR (1 mg/kg, ip) groups (AC + SR groups); (D) CZ (1 mg/kg, ip) plus SR (1 mg/kg, ip) groups (CZ + SR groups).**P < 0.01: significant attenuation as compared with the control group; ++P < 0.01: significant increase as compared with the NC, IM, or NC-IM group without any cotreatments; $$P < 0.01: significant attenuation as compared with the IM group without any cotreatments; ##P < 0.01: significant attenuation as compared with the NC, IM, or NC-IM group cotreated with the efficacious HDAC inhibitor, CB1 agonist, or TRPV1 antagonist.
impairment-like effects were induced by NC.In spite of the mitigating effects of NC against stressor-induced emotional and cognitive dysfunction in certain treatment conditions accompanied by a protective influence on the relevant stress-related neurotransmitter systems, biochemical responses and synaptic plasticity, [65][66][67][68] synergistic behavioural impairments like those reported in some studies 25,38,69,70 were observed.Here, excessive augmentation in the activity of the hypothalamic-pituitary-adrenal (HPA) axis and immediate early genes associated with neural circuit mechanisms 69,71,72 may be implicated.
4][75][76][77] Several studies have reported the favourable influence of HDAC inhibitor-induced histone acetylation in the control of stress-related neural circuits. 78,791][82] Although the enhancement of Counteraction caused by the TRPV1 antagonist against attenuating effects of the CB1 antagonist on HDAC inhibitor-or CB1 agonist-induced anxiolytic-like behavioural alterations in the NC and/or IM treatment groups.The parameter values of the EPM test at the 2-h time point after the last NC (0.8 mg/kg, sc) and/or IM (10 min) treatment are shown as means with SD bars (n = 10) for each anxiolytic-like drug (HDAC inhibitor or CB1 agonist) plus CB1 antagonist cotreatment group with or without additional TRPV1 antagonist (with each ip dose [mg/kg]), and statistical significance in post hoc tests is denoted using the symbols as defined below.The detailed data and statistical results have been included in Table S4.(A) SB (100 mg/kg, ip) plus SR (1 mg/kg, ip) cotreatment groups with (or without) additional CZ (1 mg/kg, ip) (SB + SR + CZ groups); (B) VA (300 mg/kg, ip) plus SR (1 mg/kg, ip) cotreatment groups with (or without) additional CZ (1 mg/kg, ip) (VA + SR + CZ groups); (C) AC (0.2 mg/kg, ip) plus SR (1 mg/kg, ip) cotreatment groups with (or without) additional CZ (1 mg/kg, ip) (AC + SR + CZ groups).**P < 0.01: significant attenuation as compared with the control group; ++P < 0.01: significant increase as compared with the NC, IM, or NC-IM group without any cotreatments; $$P < 0.01: significant attenuation as compared with the IM group without any cotreatments; ##P < 0.01: significant attenuation as compared with the NC, IM, or NC-IM group cotreated with the efficacious (anxiolytic-like) HDAC inhibitor or CB1 agonist; &&P < 0.01: significant increase as compared with the NC, IM, or NC-IM group cotreated with the mitigating drug (HDAC inhibitor or CB1 agonist) plus SR.
stressor-like effects accompanied by impaired behavioural plasticity has been suggested at high doses of SB and VA, 83,84 controlled HDAC inhibition seems to mitigate the NC-and/or IM-induced anxiety-and working memory impairment-like behaviours.
4.2 | Effects of ECB and/or TRPV1 system-related ligands against NC-and/or IM-induced anxiety-and working memory impairment-like behavioural alterations and epigenetic interactions with HDAC inhibitors In the present study, the CB1 agonist AC and the TRPV1 antagonist CZ mitigated the NC-and/or IM-induced anxiety-like behavioural alterations.Against the working memory impairment-like behavioural alterations, mitigating effects were induced by the TRPV1 agonist OL and the CB1 antagonist SR.7][28][29][30] However, the anxiolytic-like effects of AC were limited (efficacious at 0.05-0.2mg/kg), and differential involvement of the complementary neurotransmitter systems represented by the TRPV1 system [91][92][93] could be assumed.Like AC, CZ mitigated the anxiogeniclike effects of NC and/or IM, consistent with previous experimental studies using CZ-like TRPV1 antagonists, 46,[94][95][96] and altered facilitation of ECB system-related complementary neurotransmission [97][98][99] has been suggested.Intriguingly, the anxiolytic-like effects of the   Against working memory impairment-like behaviours, unlike against anxiety-like behaviours, ameliorating effects were induced by the CB1 antagonist SR, for which combined involvement of the complementary neurotransmitter systems, [102][103][104] along with specific biochemical/neurophysiological modifications in memory-related brain regions, 105,106 could be assumed.Such mitigating effects were also exerted by the TRPV1 agonist OL, consistent with previous studies using TRPV1 agonists, 107,108 and simultaneous modulation of ECB system-related complementary neurotransmission observed in anxiety-related behavioural plasticity [97][98][99] seemed to be implicated.
0][111] Although the detailed mechanisms underlying the discrepancy in the efficacy of CB1/TRPV1 ligands between the anxiety-and working memory impairment-related behaviours remain elusive, the differences in the relevant brain regions, [112][113][114][115] along with polymodal regulation of synaptic plasticity involved in the two neurotransmitter systems, 116,117 seemed to contribute, and this may be controlled subtly at the epigenetic level. .**P < 0.01: significant attenuation as compared with the control group; ++P < 0.01: significant increase as compared with the NC, IM, or NC-IM group without any cotreatments; @@P < 0.01: significant attenuation as compared with the NC group without any cotreatments; $$P < 0.01: significant attenuation as compared with the IM group without any cotreatments; ##P < 0.01: significant attenuation as compared with the NC, IM, or NC-IM group cotreated with the efficacious (working memory improving-like) HDAC inhibitor or TRPV1 agonist; &&P < 0.01: significant increase as compared with the NC, IM, or NC-IM group cotreated with the mitigating drug (HDAC inhibitor or TRPV1 agonist) plus CZ.

(
arachidonylcyclopropylamide [AC]), the CB1 antagonist SR 141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride [SR]), the TRPV1 agonist olvanil (OL), and the TRPV1 antagonist capsazepine (CZ), which were all purchased from Tocris Cookson Inc. (Ellisville, Missouri, USA), the data were collected and shown for the following intraperitoneal (ip) doses: 50, 100, and 200 mg/kg for SB; 200, 300, and 400 mg/kg After the number of entries into open arms, the time spent on open arms (seconds) and the total number of entries into arms were evaluated (5-min [300 s] test periods), the percentage of entries into open arms and the percentage of time spent on open arms were calculated as parameters of anxiety-related behaviours.These evaluations were performed at the 2-h time point after the last NC, IM, or NC-IM treatment.At the beginning of each experimental session, each mouse was placed diagonally in the center platform of the maze, facing both the open and enclosed arms.

3 | RESULTS 3 . 1 |
examining the NC-and/or IM-induced anxiety-and working memory-related behavioural alterations and the effects of each HDAC inhibitor, CB1 or TRPV1 ligand, a 2 Â 2 or 4 Â 4 factorial design was used.With respect to the experiments examining the interacting role of HDAC inhibition (histone acetylation) with the ECB and/or TRPV1 system, a 4 Â 2 Â 4 or 4 Â 2 Â 2 factorial design was used.For pairwise comparisons, Bonferroni post hoc tests were performed.All of the comparisons were performed using the statistical software "Excel Statistics" (Social Survey Research Information Co. Ltd., Tokyo, Japan).25,38P values less than 0.05 were considered to be statistically significant.Mitigating effects of the HDAC inhibitor, CB1 agonist, or TRPV1 antagonist against NC and/or IMinduced anxiety-like behavioural alterations in the EPM test Consistent with previous studies,25,38 anxiety-like behavioural alterations in the EPM test, that is, significantly attenuated percentage of entries into open arms and significantly attenuated percentage of time spent on open arms as compared with the control group, were induced by repeated NC, IM, or NC-IM treatments (Figure 1 and Table S1; NC, IM, or NC-IM group).For the NC-IM group, each parameter value was significantly attenuated as compared with the IM group (Figure 1 and Table S1; NC-IM group).Using two-way ANOVA, statistically significant main effects of NC (F(1, 36) = 268.59,P = 2.84 Â 10 À18 [P < 0.001] for the percentage of entries into open arms and F(1, 36) = 96.98,P = 9.34 Â 10 À12 [P < 0.001] for the percentage of time spent on open arms) and IM (F(1, 36) = 116.92,P = 7.39 Â 10 À13 [P < 0.001] for the percentage of entries into open arms and F(1, 36) = 48.32,P = 3.80 Â 10 À8 [P < 0.001] for the F I G U R E 1 Legend on next page.percentage of time spent on open arms) and statistically significant interactions between the NC and IM treatment in the NC-IM group (F (1, 36) = 118.07,P = 6.45 Â 10 À13 [P < 0.001] for the percentage of entries into open arms and F(1, 36) = 23.68,P = 2.26 Â 10 À5 [P < 0.001] for the percentage of time spent on open arms) were observed (Table gating effects, that is, recoveries from both attenuated percentage of entries into open arms and attenuated percentage of time spent on open arms, were observed in the NC, IM, and NC-IM groups cotreated with the HDAC inhibitor SB (50-200 mg/kg) or VA (200-400 mg/kg), the CB1 agonist AC (0.2-1 mg/kg), or the TRPV1 antagonist CZ (1-5 mg/kg) (Figure 1A-D and Table S1A-D).This is consistent with the results of two-way ANOVA revealing statistically significant interactions between the following combined treatments: NC and/or IM Â SB (F(9, 144) = 21.73,P = 6.85 Â 10 À23 [P < 0.001] for the percentage of entries into open arms and F(9, 144) = 5.42, P = 2.16 Â 10 À6 [P < 0.001] for the percentage of time spent on open arms), NC and/or IM Â VA (F(9, 144) = 22.12, P = 3.31 Â 10 À23 [P < 0.001] for the percentage of entries into open arms and F (9, 144) = 5.73, P = 8.85 Â 10 À7 [P < 0.001] for the percentage of time spent on open arms), NC and/or IM Â AC (F(9, 144) = 30.53,P = 2.97 Â 10 À29 [P < 0.001] for the percentage of entries into open arms and F(9, 144) = 6.58,P = 7.70 Â 10 À8 [P < 0.001] for the percentage of time spent on open arms), and NC and/or IM Â CZ (F (9, 144) = 29.13,P = 2.53 Â 10 À28 [P < 0.001] for the percentage of entries into open arms and F(9, 144) = 6.68,P = 5.78 Â 10 À8 [P < 0.001] for the percentage of time spent on open arms) (Table cotreated with the HDAC inhibitor SB (100-200 mg/kg) or VA (200-400 mg/kg), the TRPV1 agonist OL (1-2.5 mg/kg), or the CB1 antagonist SR (1-2 mg/kg) (Figure 2A-D and Table S2A-D).This is consistent with the results of two-way ANOVA revealing statistically significant main effects of SB (F(3, 144) = 12.45, P = 2.75 Â 10 À7 [P < 0.001]), VA (F(3, 144) = 13.32,P = 1.01 Â 10 À7 [P < 0.001]), OL (F(3, 144) = 12.36, P = 3.07 Â 10 À7 [P < 0.001]), and SR (F(3, 144) = 13.29,P = 1.05 Â 10 À7 [P < 0.001]) (Table the CB1 antagonist SR (0.5-2 mg/kg) were examined for the most effective dose of the HDAC inhibitor SB (100 mg/kg) or VA (300 mg/kg), as well as for the most effective dose of the CB1 agonist AC (0.2 mg/kg), to investigate the interacting roles of HDAC inhibitors with the ECB system.Against the "anxiolytic-like" effects of SB or VA, as well as against those effects of AC, significant attenuating effects of SR (1-2 mg/kg) were observed for each parameter in the NC, IM, and NC-IM groups (Figure3A-C and TableS3A-C).Moreover, even against the most anxiolytic-like dose of the TRPV1 antagonist CZ (1 mg/kg), significant attenuating effects were observed for SR (1-2 mg/kg) in the NC, IM, and NC-IM groups (Figure3Dand F I G U R E 1 Mitigating effects of the HDAC inhibitors, CB1 agonist, or TRPV1 antagonist against anxiety-like behaviours.The parameter values of the EPM test (percentages of entries into open arms and time spent on open arms) at the 2-h time point after the last NC (0.8 mg/kg, sc) and/or IM (10 min) treatment are shown as means with standard deviation (SD) bars (n = 10) for each HDAC inhibitor (SB or VA), CB1 agonist (AC), or TRPV1 antagonist (CZ) cotreatment group (with each ip dose [mg/kg]), and statistical significance in posthoc tests is denoted using the symbols as defined below.The detailed data and statistical results have been included in TableS1.(A) SB (100 mg/kg, ip) cotreatment groups (SB groups); (B) VA (300 mg/kg, ip) cotreatment groups (VA groups); (C) AC (0.2 mg/kg, ip) cotreatment groups (AC groups); (D) CZ (1 mg/kg, ip) cotreatment groups (CZ groups).**P < 0.01: significant attenuation as compared with the control group; ++P < 0.01: significant increase as compared with the NC, IM, or NC-IM group without any cotreatments; $$P < 0.01: significant attenuation as compared with the IM group without any cotreatments.

F I G U R E 3
Interacting effects between the CB1 antagonist and mitigating (anxiolytic-like) drug (i.e., HDAC inhibitor, CB1 agonist, or TRPV1 antagonist) against anxiety-like behavioural alterations caused by NC and/or IM.The parameter values of the EPM test at the 2-h time point after the last NC (0.8 mg/kg, sc) or IM (10 min) treatment are shown as means with SD bars (n = 10) for each mitigating drug plus CB1 antagonist group (with each ip dose [mg/kg]), and statistical significance in post hoc tests is denoted using the symbols as defined below.The detailed data and statistical results have been included in Table

F I G U E 5
Interacting between the TRPV1 antagonist mitigating (working memory improving-like) drug (i.e., HDAC inhibitor, TRPV1 agonist, or CB1 antagonist) against memory impairment-like behavioural alterations caused by NC and/or IM.The parameter values of the Y-maze test at the 2-h time point after the last NC (0.8 mg/kg, sc) or IM (10 min) treatment are shown as means with SD bars (n = 10) for each mitigating drug plus TRPV1 antagonist group (with each ip dose [mg/kg]), and statistical significance in post hoc tests is denoted using the symbols as defined below.The detailed data and statistical results have been included in

HAYASE
TRPV1 antagonist CZ, like the effects of the CB1 agonist AC, were attenuated by the CB1 antagonist SR.Moreover, the antianxiolyticlike effects of SR against AC were further counteracted by CZ.From these results, it can be hypothesized that the mutual control of the ECB and TRPV1 systems91,100,101 may crucially affect the manifestation of anxiety-related behavioural plasticity.
Like the effects induced by AC and CZ, the anxiolytic-like effects of the HDAC inhibitors SB and VA against the NC and/or IM treatment were significantly attenuated by the CB1 antagonist SR, which was further counteracted by the TRPV1 antagonist CZ.On the other hand, the working memory improving-like effects of the HDAC inhibitors SB and VA were significantly attenuated by CZ, which was further counteracted by SR.From such results, the involvement of HDAC inhibitor-induced histone acetylation in the interacting influence of the ECB and TRPV1 system-related ligands on the anxiogenic-and working memory impairment-like behaviours was hypothesized.The antianxiolytic-like effects of SR against SB and VA which were further counteracted by CZ, as well as the antimemory improving-like effects of CZ which were further counteracted by SR, may reflect some mediatory roles of histone acetylation in the interplay between the ECB and TRPV1 systems.A number of studies have suggested the contribution of reduced histone acetylation to both stress (or drug)-related impaired emotional/cognitive behaviours and disturbed function of either the ECB or TRPV1 system.[118][119][120][121][122][123][124]Nevertheless, few studies have discussed the role of HDAC inhibitor-induced histone acetylation in the interacting behavioural influence of the ECB and TRPV1 systems.125Recently, the cooperative epigenetic involvement of the ECB and TRPV1 systems in the pathogenesis of various diseases, including aberrant inflammatory responses observed in COVID-19, has been suggested.126,127Controlled histone acetylation may contribute to normalized crosstalk between the ECB and TRPV1 systems, which could regulate both emotional and cognitive behaviours, and develop potential therapeutic interventions.4.3 | ConclusionIn summary, the present results showed the protective effects of HDAC inhibitors (SB and VA) against the NC-and/or IM-induced anxiety-and working memory impairment-like behavioural alterations, for which the anxiolytic-like effects were mimicked by the CB1 agonist AC or the TRPV1 antagonist CZ, whereas the working memory improving-like effects were mimicked by the CB1 antagonist SR or the TRPV1 agonist OL.Moreover, a reversing influence was observed for SR against the anxiolytic-like effects of the HDAC inhibitors, AC or CZ, which was further counteracted by CZ.On the other hand, a reversing influence was observed for CZ against the working memory improving-like effects of the HDAC inhibitors, SR or OL, which was further counteracted by SR.These findings of the interrelated involvement may suggest a therapeutically important role of the HDAC inhibitor-induced epigenetic histone acetylation in the interplay between the ECB and TRPV1 systems against aberrant behavioural plasticity.F I G U R E 6 Counteraction caused by the CB1 antagonist against attenuating effects of the TRPV1 antagonist on HDAC inhibitor-or TRPV1 agonist-induced working memory improving-like behavioural alterations in the NC and/or IM treatment groups.The parameter values of the Y-maze test at the 2-h time point after the last NC (0.8 mg/kg, sc) and/or IM (10 min) treatment are shown as means with SD bars (n = 10) for each working memory improving-like drug (HDAC inhibitor or TRPV1 agonist) plus TRPV1 antagonist cotreatment group with or without additional CB1 antagonist (with each ip dose [mg/kg]), and statistical significance in post hoc tests is denoted using the symbols as defined below.The detailed data and statistical results have been included in TableS6.(A) SB (100 mg/kg, ip) plus CZ (1 mg/kg, ip) cotreatment groups with (or without) additional SR (1 mg/kg, ip) (SB + CZ + SR groups); (B) VA (300 mg/kg, ip) plus CZ (1 mg/kg, ip) cotreatment groups with (or without) additional SR (1 mg/kg, ip) (VA + CZ + SR groups); (C) OL (1 mg/kg, ip) plus CZ (1 mg/kg, ip) cotreatment groups with (or without) additional SR (1 mg/kg, ip) (OL + CZ + SR groups) impairs synaptic plasticity gene expression and behavior in mice.Int J Neuropsychopharmacol. 2020;23(5):324-338.doi:10.1093/ijnp/pyaa017 124.Wang SE, Ko SY, Jo S, et al.TRPV1 regulates stress responses through HDAC2.Cell Rep. 2017;19(2):401-412.doi:10.1016/j.celrep.2017.03.050 125.Hayase T. Epigenetic involvement of the endocannabinoid vs. endovanilloid system in anxiogenic-like effects of nicotine as a stressor, in: Abstracts of the 26th Annual Meeting of the International Behavioral Neuroscience Society.2017:83-84.Accessed June 3, 2024.https://www.ibnsconnect.org/assets/docs/2017ibnsprogram.pdf126.Hong S, Zheng G, Wiley JW.Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system.Gastroenterology.2015;148(1):148-157.e7.doi:10.1053/j.gastro.2014.09.032 127.Nagarkatti P, Miranda K, Nagarkatti M. Use of cannabinoids to treat acute respiratory distress syndrome and cytokine storm associated with coronavirus disease-2019.Front Pharmacol.2020;11:589438.doi:10.3389/fphar.2020.589438